Numerous animal studies have shown that the intravenous (iv) injection of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) can protect the heart against reversible postischemic dysfunction (myocardial stunning). The PI hypothesizes that direct gene transfer of cDNAs encoding antioxidant enzymes will similarly protect the myocardium against ischemia/reperfusion (I/R) injury. The PI proposes to test this hypothesis by using 2nd-generation adenoviral vectors (Ad5) to over-express SOD, CAT and glutathione peroxidase (GPX) in a conscious rabbit model of myocardial stunning and infarction. A multidisciplinary approach will be used to pursue the following specific aims: 1. To construct and evaluate Ad5 vectors expressing intra- and extracellular forms of SOD. Previous work by the co-investigator (Dr. Roberto Bolli) showed that a combination of SOD and catalase markedly attenuated myocardial stunning, whereas either enzyme alone was ineffective. The PI proposes that this was due to the failure of these enzyme to enter the cytosol, and that intracellular elevations of SOD alone may be cardioprotective. The PI has already created an Ad5 vector expressing the extracellular of SOD, and a similar vector will be made to express intracellular SOD. 2. To construct and evaluate Ad5 vectors expressing intra- and extracellular forms of CAT. As with SOD. the failure of iv CAT alone to protect against I/R injury may be due to its inability to enter the cytoplasm. Most reactive oxygen species (ROS) originate within cells, so CAT should be more effective if it is located intracellularly. Ad5 vectors expressing both intra- and extracellular forms of CAT will be constructed to address this unresolved issue. 3. To construct and evaluate Ad5 vectors expressing intra- and extracellular forms of GPX. GPX plays a critical role in relieving oxidative stress by neutralizing both lipid hydroperoxides and H202, but its cardioprotective potential has yet to be elucidated. The PI plans to construct Ad5 vectors that express both the intra- and extracellular forms of GPX to perform a comprehensive study of the antioxidant enzymes that could reduce myocardial I/R injury. The 2nd generation Ad5 vectors expressing each antioxidant enzyme will be functionally tested in vitro before initiating the in vivo studies. 4. To evaluate Ad5-mediated gene therapy in a conscious rabbit model of myocardial stunning and infarction. The PI is collaborating with Dr. Roberto Bolli to evaluate the antioxidant Ad5 vectors in a physiological, conscious rabbit model of myocardial I/R injury. Direct gene transfer will be compared with the iv injection of antioxidant enzymes to clarify the potentially therapeutic role of antioxidant enzymes in myocardial I/R injury, and to determine for the first time whether gene therapy can attenuate myocardial stunning and reduce infarct size in a conscious animal model.